The present invention relates to the field of microencapsulation of cells, and more specifically, to the production of microencapsulated cells.
Microencapsulation is an immunoisolation technique available for immunoprotection of cells to be transplanted, which reduces or eliminates the use of immunosuppressive drugs. Uludag H, De Vos P, Tresco P A: Technology of mammalian cell encapsulation. Adv Drug Delivery Rev 42: 29-64, 2000. Although, microencapsulation as a viable procedure to immunoisolate cells for transplantation was introduced more than twenty years ago (Lim F, Sun A M. Microencapsulated islets as bioartificial endocrine pancreas. Science 210: 908-910, 1980), it has had a slow progress towards clinical application, for example, due to slow production rates and the appearance of fibrotic overgrowths around the capsules, which can result in endotoxin contamination, e.g., oxygen and nutrient deprivation of the enclosed cells.
One of the many prospective applications of this technology is the development of a reliable bioartificial liver in the form of encapsulated hepatocytes, for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to orthotopic liver transplantation for patients with fulminant hepatic failure. Joly A, Desjardins J-F, Fredmond B, et al. Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver. Transplantation 63: 795-803, 1997.
Examples of devices for microencapsulation include the air-syringe pump droplet generator (Wolters G H, Fritschy W M, Gerrits D, Van Schilfgaarde R: A versatile alginate droplet generator applicable for microencapsulation of pancreatic islets. J Appl Biomat 3: 281-286, 1992) and the electrostatic bead generator (Hsu BR-S, Chen H-C, Fu S-H, Huang Y-Y, Huang H-S: The use of field effects to generate calcium alginate microspheres and its application in cell transplantation. J Formos Med Assoc 93: 240-245, 1994.). Each of these devices is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into spherical beads. Various methods for the production of encapsulated cells in increased numbers have been attempted, including the simultaneous production of multiple droplets in a multiple needle approach (De Vos P, De Haan B J, Schilfgaarde R. Upscaling the production of microencapsulated pancreatic islets. Biomaterials 18: 1085-1090, 1997) or increasing the number of cells/mL of alginate suspension in the syringe to increase the probability of the formation of encapsulated cells. However, air-syringe pump droplet generators or electrostatic bead generators may be incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. Moreover, a prolonged process of encapsulation of cells may adversely affect the viability of the cells.
For example, in a study with four nozzles, the nozzles are fitted to a header plate where in the cell in alginate is supplied and pushed through four hypodermic needles. De Vos P, De Haan B J , Schilfgaarde R. Upscaling the production of microencapsulated pancreatic islets. Biomaterials 18: 1085-1090, 1997. However, this may not be scaled up effectively because of the support mass surrounding the hypodermic needle such as couplings and seals, which provides a spacing of about 1 cm between needles. An increase in the number of joints in the flow path through which the cell—alginate suspension travels may result in a higher the possibility of stagnation, clogging, and contamination. Furthermore, alginate solutions used for encapsulation are viscous, making the process potentially fraught with the risk of clogging when small gauge needles are used to produce microcapsules of desirable size range (e.g., <800 microns in diameter). When needle clogging occurs, the process of unclogging the needle for resumption of encapsulation further increases the duration of microencapsulation of large batches of cells for therapeutic use. High density of parallel needles may not provide the access needed to clean clogged needles in the center of the array in a fairly dense grid. Multiple needles with a common flow header may not be viable and/or efficient.
Even using such an approach, production rates at several orders of magnitude higher may be desirable to meaningfully produce sufficient quantities of encapsulated and viable cells for transplantation in human subjects. For example, it has been estimated that for the 1 million islets needed for transplantation in a diabetic human subject, about 100 hours may be required to complete the encapsulation of this number of islets, assuming one islet/microcapsule and a single needle operation. However, in practice, it has actually been estimated that the duration of the process may be closer to 200 hours because of the additional steps involved in the encapsulation procedure, following the generation of the initial cell-containing alginate microspheres.
Moreover, it has been reported that by using the syringe method, the proportion of spheres that contain cells is only about 50%. Attempts may be made to increase the concentration of cells in the alginate suspension to increase the chance process of encapsulating a cell and thereby increasing the productivity. However, this may provide only a two-fold increase in productivity. Further, an increase in the number of cells/mL alginate could cause an increase in the number of beads of cells with imperfections, such as cell protrusion into the bead membrane. Protrusion of encapsulated tissue through the microcapsule membrane may activate the cell-mediated host immune response leading to microcapsule transplant rejection. Sun A M. O'Shea G M. Goosen M F: Injectable microencapsulated islet cells as a bioartificial pancreas. Appl Biochem Biotechnol 10: 87-99, 1984.